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Biomedical subjects

R J Walter

Publications and source records attributed to R J Walter.

53 records · Page 3Linked to original sources
PubMed1984

Hematoporphyrin derivative photoradiation therapy of the rat 9L gliosarcoma brain tumor model.

The distribution of hematoporphyrin derivative (HPD) in the rat 9L gliosarcoma intracerebral brain tumor model at 4, 24, 48, and 72 hours after intravenous administration was evaluated using a digital video fluorescence microscopy technique. Maximum tissue fluorescence in the normal brain was observed at 4 hours, whereas maximum fluorescence in the tumor regions occurred at 24 hours. Although the fluorescence counts suggested that there was significant uptake of HPD within the tumors when compared to normal brain, only 33% to 44% of each of the eight tumors surveyed showed fluorescence. In response to a laser light dose (633 nm) of 30 J/cm2, six rats that had been sensitized with HPD had a patchy coagulation necrosis involving up to 70% of the total tumor volume. In contrast, four rats given HPD only or exposed to laser only had no areas of necrosis, as observed on histological examination. In a group of 30 rats, no prolongation of survival was observed in response to photoradiation therapy.

Animals↗
PubMed1984

Mitochondrial fluorescence patterns in rhodamine 6G-stained myocardial cells in vitro. Analysis by real-time computer video microscopy and laser microspot excitation.

Cellular fluorescence in vitro has been studied employing a low light-level video system interfaced with a real-time image array-processing computer system. Changes in cytoplasmic (mitochondrial) fluorescence in myocytes employing the probe rhodamine 6G have been studied over real time with the aid of several computer-based programs. An oscillating pattern of fluorescence is observed that appears to reflect localized variations in mitochondrial activity. The low light level video computer system used in this study compares favorably with laser-stimulated microspot fluorescence photon-counting techniques for the detection of subcellular fluorescence.

Animals↗
PubMed1984

Localization of chemotactic peptide receptors on rabbit neutrophils.

The chemotaxis of blood leukocytes is initiated by the binding of a chemoattractant to specific receptors on the leukocyte cell surface. Although a great deal is known about the biochemical and morphological events accompanying chemotactic activation, there is very little morphological information about the chemoattractant receptors themselves. This latter information is needed so that we may understand the mechanism by which these inflammatory cells detect and respond to chemical gradients. One class of chemotactic factors extensively used to characterize the complex behavioral responses following leukocyte activation are the synthetic formylmethionyl peptides. These peptides, now known to be the analogs of the naturally occurring N-terminal peptides produced by bacteria, are released into culture medium and are believed to be responsible, at least in part, for the accumulation of leukocytes at the sites of bacterial infection. We have localized the receptors for the chemotactic hexapeptide N-formylnorleucyl-leucyl-phenylalanine-norleucyl-[125I]tyrosyl-lys ine [N-fNle-Leu-Phe-Nle-[125I]Tyr-Lys] on whole rabbit peritoneal neutrophils (PMN) using light microscope autoradiography. By this method, the inherent formylpeptide receptor distribution on cells incubated at 4 degrees C appears to be uniform over the surface of both rounded and structurally polarized PMN. Following a short 37 degrees C incubation, cells retain a large proportion of labelled hexapeptide at or near the cell surface and, in addition, polarized PMN redistribute the hexapeptide anteriorly away from the cell uropod.

Animals↗
PubMed1984

Computer-assisted video analysis of [3H]spiroperidol binding autoradiographs.

A computer-based video image processing system is described that quantifies the binding of the neuroleptic drug, [3H]spiroperidol, to rat forebrain sections. Adjacent sections were incubated in buffer containing [3H]spiroperidol or [3H]spiroperidol plus 1 microM (+)-butaclamol and exposed to tritium-sensitive film to produce autoradiographs of total binding or non-butaclamol-displaced ('non-specific') binding. The image processing system digitized each autoradiograph, attenuated geometric distortion and unevenness of background illumination, and reassigned the digitized image intensities (gray values) to be a linear function of fmol [3H]spiroperidol bound per mg protein by using a calibration curve generated from 3H-containing tissue standards. An image of butaclamol-displaced ('specific') [3H]spiroperidol binding was produced by subtracting the linearized image of non-specific binding from the superimposed image of total binding. An image of percent specific [3H]spiroperidol binding was obtained by dividing the image of specific binding by the superimposed image of total binding. The computer-derived images, which could be displayed in gray tones or pseudocolor-coded, revealed that the greatest amounts of specific [3H]spiroperidol binding (1000-3000 fmol/mg protein; 60-80% specifically bound) were located in layers 5A and 5C of the neocortex, the claustrum, and in the caudate-putamen, where a lateral-to-medial binding gradient occurred. [3H]Spiroperidol was bound to a lesser extent (400-1000 fmol/mg protein; 31-51% specifically bound) to the medial nucleus accumbens septi or olfactory tubercle and without measurable specificity to the lateral septum, anterior commissure, corpus callosum, or superficial neocortex. These procedures are particularly useful for the quantitative and visual analysis of autoradiographs in which [3H]ligand binding is associated with more than a single site.

Animals↗
PubMed1983

Cell motility and microtubules in cultured fibroblasts from patients with Kartagener syndrome.

Patients with Kartagener syndrome (KS) show defects in ciliary and flagellar movement that are usually associated with the partial or total absence of dynein side arms from axonemal microtubules. Dynein is essential for such movements, but its involvement in other cellular (particularly microtubule-related) processes is unknown. It has recently been reported that neutrophils from KS patients show impaired motility including responses to chemotactic stimuli, suggesting that dynein-like proteins may be generally involved in motile processes. In support of this, we have now found that spontaneous motility of cultured skin fibroblasts from KS patients is also markedly impaired. Three cell lines derived from skin explants of KS patients with deficient dynein side arms in nasal cilia and eight cell lines derived from normal volunteers were studied. Fibroblasts were seeded into dishes containing colloidal gold-coated cover glasses [Albrecht-Buehler, 1977], incubated for 24h at 37 degrees C, and the area of cell "phagokinetic" tracks determined. Each cell line studied in this manner reproducibly displayed an amount of spontaneous motility characteristic for that cell line. The mean track area (+/- SE) for all control cells studied was 14.6 +/- 0.5 X 10(3) micron2 whereas for KS fibroblasts was 8.7 +/- 0.4 X 10(3) micron2 (P less than 0.001). Immunofluorescence microscopy using antitubulin and antihuman 210 K MAP antibodies revealed no differences in the staining patterns between control and KS fibroblasts. Pinocytic rates were identical, and the complement of tubulin and major microtubule associated proteins as seen on one-dimensional SDS polyacrylamide gel autoradiographs appeared similar for control and KS cells. Thus, the observed motility defect is probably not the result of alterations in the occurrence or distribution of microtubules or in the occurrence or binding of the major microtubule-associated proteins. This defect in cellular motility may be related to the absence of dynein or may reflect another independent cellular defect.

Adenosine Triphosphatases↗
PubMed1982

Membrane activity and topography of F-Met-Leu-Phe-Treated polymorphonuclear leukocytes. Acute and sustained responses to chemotactic peptide.

The chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (f-Met-Leu-Phe) causes a dramatic stimulation of membrane ruffling and a fluid pinocytosis in polymorphonuclear leukocytes (PMNs). These responses are maximal by 1 minute and subside within 5-10 minutes. The same immediate responses characterize cells exposed to several peptide hormones and may thus represent an essential component of target cell activation by peptides. The stimulation of the whole membrane following f-Met-Leu-Phe binding is succeeded by the development of a polarized cell shape with a posterior uropod and a broad anterior lamellipodium, both subtended by microfilaments. Membrane components and functions segregate into distinct domains on polarized PMNs. Thus, succinyl concanavalin A-receptor complexes are capped and internalized by receptor-mediated endocytosis at the uropod; the uptake by fluid pinocytosis of fluorescein-dextran is restricted to the uropod; and coated pits and coated vesicles are concentrated at the uropod. The lamellipodium excludes coated pits and lacks pinocytic activity but shows preferential binding of immunoglobulin aggregates, presumably to Fc receptors. The origin and physiologic implications of these asymmetries of membrane molecular and functional topography on polarized cells are discussed.

Adsorption↗
PubMed1981

Computer-enhanced video microscopy: digitally processed microscope images can be produced in real time.

Digital processing techniques can be used to greatly enhance the available information in an optical image. Although this technology has been routinely used in many fields for a number of years, little application of digital image-processing techniques have been made toward analysis and enhancement of the types of images seen most often by the research biologist. We describe here a computer-based video microscope system that is capable of performing extensive manipulation and enhancement of microscope images in real time. The types of manipulations possible with these techniques greatly surpass the enhancement capabilities of photographic or video techniques alone. The speed and flexibility of this system enables experimental manipulation of the microscopic specimen based on its live processed image. These features greatly extend the power and versatility of the light microscope.

Animals↗
PubMed1981

Effects of 5-bromo-2'-deoxyuridine on Friend erythroleukaemia cells. II. Oxidative metabolism and enzyme content of whole cells and isolated mitochondria.

Untreated or bromodeoxyuridine (BrdUrd)-treated Friend erythroleukaemia (EL) cells from 15- and 72-h-cultures were harvested and the mitochondria were isolated by homogenization and differential centrifugation. Aliquots of the original cell suspensions or the final mitochondrial suspensions were either fixed for electron microscopy, assayed for enzyme activities, or introduced into a 1-ml Clarke oxygen electrode chamber. Whole BrdUrd-treated cells exhibited notable morphological alterations (see accompanying paper) but no effect was observed on whole-cell respiration. Morphologically, isolated mitochondria exhibited a highly condensed matrix and a greatly expanded outer compartment. Functionally, these mitochondria oxidized a variety of substrates at high state-3 (ADP-stimulated) rates (60-210 ng atoms O/min per mg protein) and displayed adequate respiratory control and ADP/O ratios. In the mitochondria isolated from BrdUrd-treated EL cells (both 15- and 72-h cultures), the state-3 oxygen consumption rates, respiratory control ratios, and ADP/O ratios generally decreased compared to their matched controls. These functional deficiencies coincided with in situ increases in the volume of the mitochondrial compartment and conspicuous (30-100%) increases in the specific and total activities of several mitochondrial enzymes. In addition, the mitochondria isolated from the 72-h group (treated and untreated) displayed immediate alterations when BrdUrd was added to the electrode chamber. Oxygen consumption rates dropped, respiratory control ratios changed moderately, and ADP/O ratios increased. BrdUrd may act both as an inhibitor of oxidative phosphorylation and perhaps as a sink for some of the high energy phosphate that is being generated. Thus, BrdUrd may exert its inhibitory effect on cell differentiation by interfering with mitochondrial function.

Animals↗
PubMed1980

Asymmetric Fc receptor distribution on human PMN oriented in a chemotactic gradient.

Phagocytosis of antibody-coated particles and immune complexes by polymorphonuclear leukocytes (PMN) and macrophages is initiated by binding to Fc receptors. While it appears that subsequent ingestion requires receptor-Fc interactions to occur along the entire periphery of the particle, the distribution of Fc receptors over the leukocyte surface is unknown. We show here that during chemotaxis the Fc receptors of human neutrophils are localized to the cell's leading edge. This restricted receptor topography coincides with cell orientation and does not require net forward movement of PMN. Thus Fc receptors are located in precisely that region of the cell most likely to first encounter opsonized particles.

Cell Compartmentation↗
PubMed1980

Ultrastructural and biochemical effects of D-penicillamine on mouse hepatocytes.

The ultrastructural and oxidative metabolic effects of D-penicillamine were examined in hepatocytes of mice fed various concentrations of the drug for periods up to 11 days. In virtually all animals studied, membranous material in the form of whorls, sheaves, and irregular networks was observed in some bile canaliculi. In several mice a few mitochondria were substantially enlarged, with diameters of 4-6 micron. In other animals some mitochondria became quite elongated, measuring 10 micron in length but only 0.5 micron in width. Certain mitochondria had small deposits of dense material in their outer compartment. Lysosomes contained numerous small dense particles; aggregates of these particles were also observed free in the cytosol. Despite these alterations, the vast majority of hepatocytes showed no changes whatsoever. Study of isolated hepatic mitochondria derived from the experimental animals showed no alterations in oxidative metabolism. It may be concluded that, unlike other copper-chelating agents, D-penicillamine has little significant morphological and biochemical effect on mouse hepatocytes, even when its concentration exceeds standard clinical dosages.

Animals↗
PubMed1980

Polarization of endocytosis and receptor topography on cultured macrophages.

In the 1774.2 macrophage cell line, microtubule disassembly by colchicine causes the polarization of membrane functions ane structure. Colchicine-treated cells develop a bulge or protuberance that is bordered by microvillous membrane. The protuberance is the site of concanavalin A cap formation. The fluid pinocytosis of horseradish peroxidase and of fluorescein- and rhodamine-conjugated high molecular-weight dextrans, the adsorptive pinocytosis of concanavalin A, and the concentration and phagocytosis at 37 degrees C of a range of phagocytic particles (IgG- and complement-opsonized erythrocytes, complement-opsonized zymosan, latex shpres, albumin-stabilized oil droplets) are all similarly restricted to the protuberance. A reduction in the rate of dextran pinocytosis, determined by fluorimetry, and reductions in phagocytic rates for oil emulsion and IgG-opsonized erythrocytes accompany the polarization of endocytic activity in colchicine-trated 1774.2 macrophages. Membrane receptors for phagocytic particles are not confined to the protuberance but rather may display their own unique topographical asymmetry. The inherent topography of receptors was inferred from particle distribution under conditions that limit particle-receptor redistribution (after labeling at 4 degrees C or a very brief incubation at 37 degrees C). Under these restrictive conditions, latex binding sites were detected over the whole membrane whereas receptors for IgG-opsonized erythrocytes, aggregated IgG, complement-opsonized erythrocytes, and complement-opsonized zymosan were excluded from the protuberance. Thus, functional (endocytosis) and structural (inherent receptor distribution) analyses of membrane topography define different patterns of asymmetry in protuberant cells. The asymmetry induced in 1774.2 macrophages by colchicine is highly analogous to the functional and structural polarity of epithelial cells. Exploration of this analogy may provide insight into the development of polarized epithelia and, more generally, into mechanisms by which specialized areas of membrane are established.

Animals↗
PubMed1978

Surface functions during Mitosis I: phagocytosis, pinocytosis and mobility of surface-bound Con A.

Phagocytosis, pinocytosis and the surface distribution of concanavalin A (ConA) have been analyzed during mitosis in several mammalian cell lines. Use of the bisbenzimidazole dye, Hoechst 33258, for chromosome staining after gentle fixation made possible the rapid identification and correlation of mitotic phase with surface properties. Phagocytosis of both opsonized and nonopsonized particles is markedly depressed in mitotic cells of the mouse macrophage cell line J774.1. The uptake of opsonized particles (IgG-coated erythrocytes) is impaired from early prophase through early G1, whereas phagocytosis of nonopsonized particles (latex beads) is restored by telophase. Fluid pinocytosis, determined by the uptake of soluble horseradish peroxidase, is also inhibited during mitosis. Thus peroxidase-containing cytoplasmic vesicles were virtually absent from mid-prophase through telophase in both J774 and Chinese hamster ovary (CHO) cells. Adsorptive pinocytosis of ConA was determined from the different distributions of fluorescence in single cells incubated at 37 degrees C with rhodamine-conjugated ConA (surface and cytoplasmic label), then fixed and further incubated with fluorescein-conjugated anti-ConA (surface only). The separate fluorescence of Hoechst, fluorescein and rhodamine could be optically isolated. In interphase J774 cells, ConA is rapidly internalized into cytoplasmic vesicles. In contrast, ConA is restricted to the plasma membrane from mid-prophase through telophase. In CHO, the depressed pattern of internalization is not fully established until metaphse. The surface distribution of ConA also varied dramatically as a function of mitotic phase. Between mid-prophase and early anaphase, the pattern of surface ConA-receptor complexes is diffuse. Once the cleavage furrow begins to develop, however, ConA moves into the region of the furrow. This was shown in J774, CHO and 3T3 mouse embryonic fibroblasts, and is probably universal. ConA movement into the membrane that overlies the microfilaments of the contractile ring is analogous to similar movements that occur in interphase cells during ConA cap formation and during the development of phagocytic pseudopods. The analogy emphasizes the common functional consequences of microfilament-membrane organization. It is evident that membrane processes which depend upon endocytosis--for example, certain hormone-induced signals--may be interrupted during mitosis. Inhibition of endocytosis thus may be a significant element in the control of cellular activities during mitosis and a strong influence on the properties of the emergent post-mitotic cell.

Cell Cycle↗
PubMedDate not supplied

Hemodynamic effects of delayed initiation of antioxidant therapy (beginning two hours after burn) in extensive third-degree burns.

The hemodynamic effects of the delayed initiation of antioxidant therapy with high-dose vitamin C were studied in 12 guinea pigs with third-degree burns over 70% of their body surface area. All animals were resuscitated with Ringer's lactate solution (RL) according to the Parkland formula (4 ml/kg/% burn during the first 24 hours) from 1/2 to 2 hours after burn, and the infusion rate was reduced thereafter to 25% of that of the Parkland formula. The vitamin C group (n = 6) received RL with vitamin C (14 mg/kg/hr), and the control group (n = 6) received RL only. The 24-hour fluid intake for each group was 32.5% of the Parkland formula volume. Burn wound edema in the vitamin C group was significantly less than that in the control group. The vitamin C group maintained adequate hemodynamic stability as determined with hematocrit and cardiac output values, but the control group did not. Even though the initiation of the vitamin C administration is delayed until 2 hours after burn, the hourly infusion rate of the resuscitation fluid can be reduced to 25% once it is started. Thus antioxidant therapy with adjuvant vitamin C administration may be applicable to the clinical setting in which a patient with burns arrives at the burn care facility a few hours after the burn injury occurred.

Analysis of Variance↗
PubMedDate not supplied

Xenogeneic acellular dermal matrix as a dermal substitute in rats.

Acellular dermal matrix (ADM) has been used as a dermal substitute for the treatment of deep burns, but the availability of cadaver skin for the production of ADM is limited. The usefulness of porcine ADM as a xenogeneic dermal substitute in rats was studied. With the use of Dispase II (Boehringer Mannheim, Indianapolis, Ind) and Triton X-100 (US Biochemicals, Cleveland, Ohio), xenogeneic ADM was prepared from commercially available, cryopreserved porcine skin, and allogeneic ADM from the rats was also prepared. Four full-thickness injuries 225 mm2 in size were created on the dorsum of each rat. One of these wounds was treated with xenogeneic ADM and 1 was treated with allogeneic ADM, and immediately a 0.005-in thick split-thickness skin graft was placed over the ADM. The other 2 wounds were covered with 0.005- or 0.017-in thick split-thickness skin grafts alone. The wounds were evaluated macro- and microscopically 10, 14, 20, and 30 days after grafting. At 30 days after grafting, contraction of the wounds that contained xenogeneic ADM was significantly greater than that of the wounds that contained allogeneic ADM. Graft take was poor in the wounds that contained xenogeneic ADM at 14 days after surgery and moderately good in those that contained allogeneic ADM. The use of thick autografts resulted in the best wound healing, whereas the use of thin autografts resulted in considerable wound contraction. Allogeneic ADM diminished this contraction, but wound healing was significantly worsened when xenogeneic ADM was used.

Animals↗